Assay for type I collagen carboxy-terminal telopeptide analytes

ABSTRACT

A method of determining collagen degradation in vivo, comprising quantitating the concentration of a peptide in a body fluid, the peptide being a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link or a type III collagen telopeptide containing a hydroxylysyl pyridinoline cross-link. The method includes immunometric assays, fluorometric assays, and electrochemical titrations for quantitation. The structures of specific peptides having cross-links and kits for quantitating these peptides in a body fluid are described.

This invention was made with government support under one or more grantsAM 26489, AR 37318, AM 30774, and AR 36794 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

This is a continuation of prior application Ser. No. 08/537,502, filedOct. 2, 1995, which is a continuation of application Ser. No.08/226,070, filed Apr. 11, 1994 (U.S. Pat. No. 5,455,179), which in turnis a continuation of application Ser. No. 07/823,270, filed Jan. 16,1992 (U.S. Pat. No. 5,532,169), which in turn is a divisional of priorapplication Ser. No. 07/444,881, filed Dec. 1, 1989 (U.S. Pat. No.5,140,103 ), which in turn is a continuation-in-part of application Ser.No. 07/118,234, filed Nov. 6, 1987 (U.S. Pat. No. 4,973,666), thebenefits of the filing dates of which are claimed under 35 U.S.C. § 120.

FIELD OF THE INVENTION

The present invention relates to methods for detecting and monitoringcollagen degradation in vivo. More specifically, it relates to methodsfor quantitating cross-linked telopeptides produced in vivo upondegradation of collagen types II and III.

BACKGROUND OF THE INVENTION

Three known classes of collagens have been described to date. The ClassI collagens, subdivided into types I, II, III, V, and XI, are known toform fibrils. These collagens are all synthesized as procollagenmolecules, made up of N-terminal and C-terminal propeptides, which areattached to the core collagen molecule. After removal of thepropeptides, which occurs naturally in vivo during collagen synthesis,the remaining core of the collagen molecule consists largely of atriple-helical domain having terminal telopeptide sequences which arenontriple-helical. These telopeptide sequences have an importantfunction as sites of intermolecular cross-linking of collagen fibrilsextracellularly.

The present invention relates to methods of detecting collagendegradation based on assaying for particular cross-linked telopeptidesproduced in vivo upon collagen degradation. In the past, assays havebeen developed for monitoring degradation of collagen in vivo bymeasuring various biochemical markers, some of which have beendegradation products of collagen. For example, bone turnover associatedwith Paget's disease has been monitored by measuring small peptidescontaining hydroxyproline, which are excreted in the urine followingdegradation of bone collagen. Russell et al., Metab. Bone Dis. and Rel.Res. 4 and 5, 255-262 (1981); and Singer, F. R., et al., Metabolic BoneDisease, Vol. II (eds. Avioli, L. V. and Kane, S. M.), 489-575 (1978),Academic Press, New York.

Other researchers have measured the cross-linking compound pyridinolinein urine as an index of collagen degradation in joint disease. See, forbackground and for example, Wu and Eyre, Biochemistry, 23:1850 (1984);Black et al., Annals of the Rheumatic Diseases, 48:641-644 (1989);Robins et al.; Annals of the Rheumatic Diseases, 45:969-973 (1986); andSeibel et al., The Journal of Rheumatology, 16:964 (1989). In contrastto the present invention, some prior researchers have hydrolyzedpeptides from body fluids and then looked for the presence of individualhydroxypyridinium residues. None of these researchers has reportedmeasuring a telopeptide containing a cross-link that is naturallyproduced in vivo upon collagen degradation, as in the present invention.

U.K. Patent application GB 2,205,643 reports that the degradation oftype III collagen in the body is quantitatively determined by measuringthe concentration of an N-terminal telopeptide from type III collagen ina body fluid.

There are a number of reports indicating that collagen degradation canbe measured by quantitating certain procollagen peptides. The presentinvention involves telopeptides rather than propeptides, the two beingdistinguished by their location in the collagen molecule and the timingof their cleavage in vivo. See U.S. Pat. No. 4,504,587; U.S. Pat. No.4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984);Niemela, Clin. Chem., 31/8:1301-1304 (1985); and Rohde et al., EuropeanJournal of Clinical Investigation, 9:451-459 (1979).

U.S. Pat. No. 4,778,768 relates to a method of determining changesoccurring in articular cartilage involving quantifying proteoglycanmonomer or antigenic fragments thereof in a synovial fluid sample. Thispatent does not relate to detecting cross-linked telopeptides derivedfrom degraded collagen.

Dodge, J. Clin. Invest., 83:647-661 (1981) discloses methods foranalyzing type II collagen degradation utilizing a polyclonal antiserumthat specifically reacts with unwound alpha-chains and cyanagenbromide-derived peptides of human and bovine type II collagens. Thepeptides involved are not cross-linked telopeptides as in the presentinvention.

Amino acid sequences of human type lIl collagen, human proα1(II)collagen, and the entire preproα1(III) chain of human type III collagenand corresponding cDNA clones have been investigated and determined byseveral groups of researchers. See Loidl et al., Nucleic Acids Research12:9383-9394 (1984); Sangiorgi et at., Nucleic Acids Research,13:2207-2225 (1985); Baldwin et al. Biochem. J., 262:521-528 (1989); andAla-Kokko et al., Biochem. J., 260:509-516 (1989). None of thesereferences specifies the structures of particular telopeptidedegradation products that could be measured to determine the amount ofdegraded fibrillar collagen in vivo.

In spite of the above-described background information, there remains aneed for effective and simple assays for determining collagendegradation in vivo. Such assays could be used to detect and monitordisease states in humans, such as osteoarthritis (type II collagendegradation), and various inflammatory disorders, such as vasculitissyndrome (type III collagen degradation).

Assays for type I collagen degradation, described in the parentapplication, U.S. Ser. No. 118,234, can be utilized to detect and assessbone resorption in vivo. Detection of bone resorption may be a factor ofinterest in monitoring and detecting diseases such as osteoporosis.Osteoporosis is the most common bone disease in man. Primaryosteoporosis, with increased susceptibility to fractures results from aprogressive net loss of skeletal bone mass. It is estimated to affect15-20 million individuals in the United States. Its basis is anage-dependent imbalance in bone remodeling, i.e., in the rates ofsynthesis and degradation of bone tissue.

About 1.2 million osteoporosis-related fractures occur in the elderlyeach year including about 538,000 compression fractures of the spine,about 227,000 hip fractures and a substantial number of early fracturedperipheral bones. Twelve to 20% of the hip fractures are fatal becausethey cause severe trauma and bleeding, and half of the survivingpatients require nursing home care. Total costs fromosteoporosis-related injuries now amount to at least $7 billion annually(Barnes, O. M., Science, 236:914 (1987)).

Osteoporosis is most common in postmenopausal women who, on average,lose 15% of their bone mass in the 10 years after menopause. Thisdisease also occurs in men as they get older and in young amenorrheicwomen athletes. Despite the major, and growing, social and economicconsequences of osteoporosis, no method is available for measuring boneresorption rates in patients or normal subjects. A major difficulty inmonitoring the disease is the lack of a specific assay for measuringbone resorption rates.

Methods for assessing bone mass often rely on measuring whole-bodycalcium by neutron activation analysis or mineral mass in a given boneby photon absorption techniques. These measurements can give onlylong-term impressions of whether bone mass is decreasing. Measuringcalcium balances by comparing intake with output is tedious, unreliableand can only indirectly appraise whether bone mineral is being lost overthe long term. Other methods currently available for assessing decreasedbone mass and altered bone metabolism include quantitative scanningradiometry at selected bone locations (wrist, calcaneus, etc.) andhistomorphometry of iliac crest biopsies. The former provides a crudemeasure of the bone mineral content at a specific site in a single bone.Histomorphometry gives a semi-quantitative assessment of the balancebetween newly deposited bone seams and resorbing surfaces.

A urinary assay for the whole-body output of degraded bone in 24 hourswould be much more useful. Mineral studies (e.g., calcium balance)cannot do this reliably or easily. Since bone resorption involvesdegradation of the mineral and the organic matrix, a specificbiochemical marker for newly degraded bone products in body fluids wouldbe the ideal index. Several potential organic indices have been tested.For example, hydroxyproline, an amino acid largely restricted tocollagen, and the principal structural protein in bone and all otherconnective tissues, is excreted in urine. Its excretion rate is known tobe increased in certain conditions, notably Paget's disease, a metabolicbone disorder in which bone turnover is greatly increased, as pointedout above. For this reason, urinary hydroxyproline has been usedextensively as an amino acid marker for collagen degradation. Singer, F.R., et al. (1978), cited hereinabove.

U.S. Pat. No. 3,600,132 discloses a process for determination ofhydroxyproline in body fluids such as serum, urine, lumbar fluid andother intercellular fluids in order to monitor deviations in collagenmetabolism. In particular, this inventor notes that in pathologicconditions such as Paget's disease, Marfan's syndrome, osteogenesisimperfecta, neoplastic growth in collagen tissues and in various formsof dwarfism, increased collagen anabolism or catabolism as measured byhydroxyproline content in biological fluids can be determined. Thisinventor measures hydroxyproline by oxidizing it to a pyrrole compoundwith hydrogen peroxide and N-chloro-p-toluenesulphonamide followed bycolorimetric determination in p-dimethyl-amino-benzaldehyde.

In the case of Paget's disease, the increased urinary hydroxyprolineprobably comes largely from bone degradation; hydroxyproline, however,generally cannot be used as a specific index. Much of the hydroxyprolinein urine may come from new collagen synthesis (considerable amounts ofthe newly made protein are degraded and excreted without ever becomingincorporated into tissue fabric), and from turnover of certain bloodproteins as well as other proteins that contain hydroxyproline.Furthermore, about 80% of the free hydroxyproline derived from proteindegradation is metabolized in the liver and never appears in the urine.Kiviriko, K. I. Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss,P. H. and Klein, L., J. Clin. Invest. 48:1 (1969).

Hydroxylysine and its glycoside derivatives, both peculiar tocollagenous proteins, have been considered to be more accurate thanhydroxyproline as markers of collagen degradation. However, for the samereasons described above for hydroxyproline, hydroxylysine and itsglycosides are probably equally non-specific markers of bone resorption.Krane, S. M. and Simon, L. S. Develop. Biochem., 22:185 (1981).

In addition to amino acids unique to collagen, various non-collagenousproteins of bone matrix such as osteocalcin, or their breakdownproducts, have formed the basis of immunoassays aimed at measuring bonemetabolism. Price, P. A. et al. J. Clin. Invest., 66:878 (1980), andGundberg, C. M. et al., Meth. Enzymol., 107:516 (1984). However, it isnow clear that bone-derived non collagenous proteins, though potentiallya useful index of bone metabolic activity are unlikely, on their own, toprovide quantitative measures of bone resorption. The concentration inserum of osteocalcin, for example:, fluctuate quite widely both normallyand in metabolic bone disease. Its concentration is elevated in statesof high skeletal turnover but it is unclear whether this results fromincreased synthesis or degradation of bone. Krane, S. M., et al.,Develop. Biochem., 22:185 (1981), Price, P. A. et al., J. Clin. Invest.,66:878 (1980); and Gundberg, C. M. et al., Meth. Enzymol., 107:516(1984).

COLLAGEN CROSS-LINKING

The polymers of most genetic types of vertebrate collagen require theformation of aldehyde-mediated cross-links for normal function. Collagenaldehydes are derived from a few specific lysine or hydroxylysineside-chains by the action of lysyl oxidase. Various di-, tri- andtetrafunctional cross-linking amino acids are formed by the spontaneousintra- and intermolecular reactions of these aldehydes within the newlyformed collagen polymers; the type of cross-linking residue variesspecifically with tissue type (see Eyre, D. R. et al., Ann. Rev.Biochem., 53:717-748 (1984)).

Two basic pathways of cross-linking can be differentiated for the banded(67 nm repeat) fibrillar collagens, one based on lysine aldehydes, theother on hydroxylysine aldehydes. The lysine aldehyde pathway dominatesin adult skin, cornea, sclera, and rat tail tendon and also frequentlyoccurs in other soft connective tissues. The hydroxylysine aldehydepathway dominates in bone, cartilage, ligament, most tendons and mostinternal connective tissues of the body, Eyre, D. R. et al. (1974) vidasupra. The operating pathway is governed by whether lysine residues arehydroxylated in the telopeptide sites where aldehyde residues will laterbe formed by lysyl oxidase (Barnes, M. J. et al., Biochem. J., 139:461(1974)).

The chemical structure(s) of the mature cross-linking amino acids on thelysine aldehyde pathway are unknown, but hydroxypyridinium residues havebeen identified as mature products on the hydroxylysine aldehyde route.On both pathways and in most tissues the intermediate,borohydride-reducible cross-linking residues disappear as the newlyformed collagen matures, suggesting that they are relatively short-livedintermediates (Bailey, A. J. et al., FEBS Lett., 16:86 (1971)).Exceptions are bone and dentin, where the reducible residues persist inappreciable concentration throughout life, in part apparently becausethe rapid mineralization of the newly made collagen fibrils inhibitsfurther spontaneous cross-linking interactions (Eyre, D. R., In: TheChemistry and Biology of Mineralized Connective Tissues, (Veis, A. ed.)pp. 51-55 (1981), Elsevier, New York, and Walters, C. et al., Calc.Tiss. Intl., 35:401-405 (1983)).

Two chemical forms of 3-hydroxypyridinium cross-link have beenidentified (Formula I and II). Both compounds are naturally fluorescent,with the same characteristic excitation and emission spectra (Fujimoto,D. et al. Biochem. Biophys. Res. Commun., 76:1124 (1977), and Eyre, D.R., Develop. Biochem., 22:50 1981)). These amino acids can be resolvedand assayed directly in tissue hydrolysates with good sensitivity usingreverse phase HPLC and fluorescence detection. Eyre, D. R. et al.,Analyte. Biochem., 137:380-388 (1984). It should be noted that thepresent invention involves quantitating particular peptides rather thanamino acids. ##STR1##

In growing animals, it has been reported that these mature cross-linksmay be concentrated more in an unmineralized fraction of bone collagenthan in the mineralized collagen (Banes, A. J., et al., Biochem.Biophys. Res. Commum., 113:1975 (1983). However, other studies on youngbovine or adult human bone do not support this concept, Eyre, D. R., In:The Chemistry and Biology of Mineralized Tissues (Butler, W. T. ed.) p.105 (1985), Ebsco Media Inc., Birmingham, Ala.

The presence of collagen hydroxypyridinium cross-kinks in human urinewas first reported by Gunja-Smith and Boucek (Gunja-Smith, Z. andBoucek, R. J., Biochem J., 197:759-762 (1981)) using lengthy isolationprocedures for peptides and conventional amino acid analysis. At thattime, they were aware only of the HP form of the cross-link. Robins(Robins, S. P., Biochem J., 207:617-620 (1982) has reported anenzyme-linked immunoassay to measure HP in urine, having raisedpolyclonal antibodies to the free amino acid conjugated to bovine serumalbumin. This assay is intended to provide an index for monitoringincreased joint destruction that occurs with arthritic diseases and isbased, according to Robins, on the finding that pyridinoline is muchmore prevalent in cartilage than in bone collagen.

In more recent work involving enzyme-linked immunoassay, Robins reportsthat lysyl pyridinoline is unreactive toward antiserum to pyridinolinecovalently linked to bovine serum albumin (Robins et al., Ann. Rheum.Diseases, 45:969-973 (1986)). Robins' urinary index for cartilagedestruction is based on the discovery that hydroxylysyl pyridinoline,derived primarily from cartilage, is found in urine at concentrationsproportional to the rate of joint cartilage resorption (i.e.,degradation). In principle, this index could be used to measure wholebody cartilage loss; however, no information on bone resorption would beavailable.

A need therefore exists for a method that allows the measurement ofwhole-body bone resorption rates in humans. The most useful such methodwould be one that could be applied to body fluids, especially urine. Themethod should be sensitive, i.e., quantifiable down to 1 picomole andrapidly measure 24-hour bone resorption rates so that the progress ofvarious therapies (e.g., estrogen) can be assessed.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of the presence ofparticular cross-linked telopeptides in body fluids of patients andnormal human subjects. These telopeptides are produced in vivo duringcollagen degradation and remodeling. The term "telopeptides" is used ina broad sense herein to mean cross-linked peptides having sequences thatare associate with the telopeptide region of, e.g., type II and type IIIcollagens and which may have cross-linked to them a residue or peptideassociated with the collagen triple-helical domain. Generally, thetelopeptides disclosed herein will have fewer amino acid residues thanthe entire telopeptide domains of type II and type III collagens.Typically, the telopeptides of the present invention will comprise twopeptides linked by a pyridinium cross-link and further linked by apyridinium cross-link to a residue or peptide of the collagentriple-helical domain. Having disclosed the structures of thesetelopeptides herein, it will be appreciated by one of ordinary skill inthe art that they may also be produced other than in vivo, e.g.,synthetically. These peptides will generally be provided in purifiedform, e.g., substantially free of impurities, particularly otherpeptides.

The present invention also relates to methods for determining in vivodegradation of type II and type III collagens. The methods involvequantitating in a body fluid the concentration of particulartelopeptides that have a 3-hydroxypyridinium cross-link and that arederived from collagen degradation. The methods disclosed in the presentinvention are analogous to those previously disclosed in U.S. Ser. No.118,234, filed Nov. 6, 1987, for determining the absolute rate of boneresorption in vivo. Those methods involved quantitating in a body fluidthe concentration of telopeptides having a 3-hydroxypyridiniumcross-link derived from bone collagen resorption.

In a representative assay, the patient's body fluid is contacted with animmunological binding partner specific to a telopeptide having a3-hydroxypyridinium cross-link derived from type II or type IIIcollagen. The body fluid may be used as is or purified prior to thecontacting step. This purification step may be accomplished using anumber of standard procedures, including cartridge adsorption andelution, molecular sieve chromatography, dialysis, ion exchange, aluminachromatography, hydroxyapatite chromatography, and combinations thereof.

Other representative embodiments of quantitating the concentration ofpeptide fragments having a 3-hydroxypyridinium cross-link in a bodyfluid include electrochemical titration, natural fluorescencespectroscopy, and ultraviolet absorbance. Electrochemical titration maybe conducted directly upon a body fluid without further purification.However, when this is not possible due to excessive quantities ofcontaminating substances, the body fluid is first purified prior to theelectrochemical titration step. Suitable methods for purification priorto electrochemical detection include dialysis, ion exchangechromatography, alumina chromatography, molecular sieve chromatography,hydroxyapatite chromatography and ion exchange absorption and elution.

Fluorometric measurement of a body fluid containing a3-hydroxypyridinium cross-link is an alternative way of quantitatingcollagen degradation (and, hence, bone resorption, if type I peptidesare quantitated). The fluorometric assay can be conducted directly on abody fluid without further purification. However, for certain bodyfluids, particularly urine, it is preferred that purification of thebody fluid be conducted prior to the fluorometric assay. Thispurification step consists of dialyzing an aliquot of a body fluid suchas urine against an aqueous solution thereby producing partiallypurified peptide fragments retained within the nondiffusate (retentate).The nondiffusate is then lyophilized, dissolved in an ion pairingsolution and adsorbed onto an affinity chromatography column. Thechromatography column is washed with a volume of ion pairing solutionand, thereafter, the peptide fragments are eluted from the column withan eluting solution. These purified peptide fragments may then behydrolyzed and the hydrolysate resolved chromatographically.Chromatographic resolution may be conducted by either high-performanceliquid chromatography or microbore high performance liquidchromatography.

The invention includes peptides having structures identical to peptidesderived from collagen degradation, substantially free from other humanpeptides, which may be obtained from a body fluid. The peptides containat least one 3-hydroxypyridinium cross-link, in particular, a lysylpyridinoline cross-link or a hydroxylysyl pyridinoline cross-link, andare derived from the telopeptide region of type II or type III collagenlinked to one or more residues from a triple-helical domain, typicallyby the action of endogenous proteases and/or peptidases.

The structures of the type II and type III telopeptides are disclosedbelow. Information on the type I telopeptides, originally presented inU.S. Ser. No. 118,234, is also included.

Structure of Cross-Linked Telopeptides Derived from Type I Collagen

A specific telopeptide having a 3-hydroxypyridinium cross-link derivedfrom the N-terminal (amino-terminal) telopeptide domain of bone type Icollagen has the following amino acid sequence: ##STR2## is hydroxylysylpyridinoline or lysyl pyridinoline, and Gln is glutamine or pyrrolidinecarboxylic acid.

The invention also encompasses a peptide containing at least one3-hydroxypyridinium cross-link derived from the C-terminal(carboxy-terminal) telopeptide domain of bone type I collagen. TheseC-terminal telopeptide sequences are cross-linked with either lysylpyridinoline or hydroxylysyl pyridinoline. An example of such a peptidesequence is represented by the formula: ##STR3## is hydroxylysyl orlysyl pyridinoline.

Since the filing of U.S. Ser. No. 118,234, the inventor has discoveredevidence of two additional type I collagen telopeptides in body fluids,having the following structures: ##STR4## These telopeptides may also bequantitated in body fluids in accordance with the present invention.

Structure of a Cross-Linked Telopeptide Derived from Type lI Collagen

A specific telopeptide having a hydroxylysyl pyridinoline cross-linkderived from the C-terminal telopeptide domain of type II collagen hasthe following amino acid sequence (referred to hereinbelow as the corepeptide structure): ##STR5## wherein the cross-linking residue depictedas Hyl-Hyl-Hyl is hydroxylysyl pyridinoline (HP), a natural3-hydroxypyridinium residue present in mature collagen fibrils ofvarious tissues.

Structure of Cross-Linked Telopeptides Derived from Type III Collagen

By analogy to the above disclosure, cross-linked peptides that arederived from proteolysis of human type III collagen may be present inbody fluids. These peptides have a core structure embodied in thefollowing parent structures: ##STR6## is hydroxylysyl or lysylpyridinoline, and Gln is glutamine or pyrrolidine carboxylic acid.

A likely cross-linked peptide derived from type III collagen in bodyfluid has the core structure: ##STR7## that is derived from twoα1(III)N-telopeptide domains linked to an hydroxylysyl pyridinolineresidue (Hyl-Hyl-Hyl).

A second possible fragment of the C-telopeptide cross-linking domain,based on the collagen types I and II peptides observed in urine, has thecore structure: ##STR8## Smaller and larger versions (differing by oneto three amino acids on each component chain) of these two peptidescorresponding to the parent sequences shown above (FORMULAE VIII and IX)may also be present and measurable in body fluids. Analogous smaller andlarger versions of each of the peptides disclosed herein form part ofthe present invention as well.

The invention includes fused cell hybrids (hybridomas) that producemonoclonal antibodies specific for the above-described collagen peptideshaving 3-hydroxypyridinium cross-links.

The invention further includes monoclonal antibodies produced by thefused cell hybrids, and those antibodies (as well as binding fragmentsthereof, e.g. F_(ab)) coupled to a detectable marker. Examples ofdetectable markers include enzymes, chromophores, fluorophores,coenzymes, enzyme inhibitors, chemiluminescent materials, paramagneticmetals, spin labels and radioisotopes.

The invention also includes test kits useful for quantitating the amountof peptides having 3-hydroxypyridinium cross-links derived from collagendegradation in a body fluid. The kits may comprise a monoclonal antibodyspecific for a peptide derived from degraded collagen and containing atleast one 3-hydroxypyridinium cross-link. The monoclonal antibodies ofthe test kits may be coupled to detectable markers such as describedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of type II collagen and a proposal for the sourceof telopeptides. It is not established whether the two telopeptidesshown come from one collagen molecule as depicted in FIG. 1 or from twocollagen molecules.

FIG. 2 shows relative fluorescence (297 nm excitation; 390 nm emission)versus fraction number (4 ml), obtained during molecular sievechromatographic purification of cross-linked telopeptides. Cross-linkedtype II collagen telopeptides are contained in the fractions designatedII.

FIG. 3A shows relative fluorescence (330 nm excitation, 390 nm emission)versus elution time of fractions during ion exchange HPLC (DEAE-5PW).Cross-linked type lI collagen telopeptides are contained in the fractiondesignated IV.

FIG. 3B shows absorbance (220 nm) versus elution time in minutes for thesame chromatogram.

FIG. 4A shows relative fluorescence (297 nm excitation, 390 nm emission)versus elution time of fractions during reverse phase HPLC. Cross-linkedtype II collagen telopeptides are eluted as indicated. The fractionsindicated by the bar (--) show evidence by sequence and compositionanalysis of the peptides indicated that retain or have lost the gly (G)and pro (P) residues.

FIG. 4B shows absorbance (220 nm) as a function of elution time duringreverse phase HPLC.

FIG. 5 compares the concentration of HP and LP in both cortical andcancellous human bone with age.

FIG. 6 depicts the cross-link molar ratios of HP to LP as a function ofage.

FIG. 7A shows relative fluorescence (297 nm excitation, >370 nmemission) as a function of elution volume during reverse phase HPLCseparation of cross-linked type I collagen N-telopeptides.

FIG. 7B shows relative fluorescence (297 nm excitation, >370 nmemission) versus elution volume during reverse phase HPLC separation ofcross-linked type I collagen C-telopeptides.

FIG. 8A shows relative fluorescence (297 nm excitation, >380 nmemission) as a function of elution time for the cross-linked type Icollagen telopeptides.

FIG. 8B shows relative fluorescence (297 nm excitation, >380 nmemission) as a function of elution time for the cross-linked type Icollagen telopeptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Type II Collagen Telopeptides

The core peptide structure of the type II collagen peptides may be foundin body fluids as a component of larger peptides that bear additionalamino acids or amino acid sequences on one or more ends of the threepeptide sequences joined by the HP residue. FIG. 1 shows how type IIcollagen telopeptides, which are linked to a triple-helical sequence,may be produced in vivo from a human source using the proteolyticenzymes pepsin and trypsin. Smaller fragments that have lost amino acidsfrom the core peptide structure, particularly from the helical sequence,may also occur in body fluids. Generally, additions or deletions ofamino acids from the core peptide structure will involve from 1 to about3 amino acids. Additional amino acids will generally be determined bythe type II collagen telopeptide sequence that occurs naturally in vivo.As examples, peptides having the following structure: ##STR9## can beisolated chromatographically from urine, and another of structure:##STR10## may also be isolated. In addition, glycosylated variants ofthe core structure and its later and smaller variants may occur in whicha galactose residue or α glucosyl galactose residue are attached to theside chain hydroxyl group of the HP cross-linking residue. Each peak inthe graph shown in FIGS. 4A and 4B may correspond to a cross-linkedfragment of particular structure that may be quantitated for purposes ofthe present invention.

These structures are consistent with their site of origin in human typeII collagen fibrils at a molecular cross-linking site formed between twoα1(II) C-telopeptides and residue 87 of a triple-helical domain, theknown sequences about which are: ##STR11##

The isolated peptide fragments represent the products of proteolyticdegradation of type II collagen fibrils within the body. The corestructure containing the HP residue is relatively resistant to furtherproteolysis and provides a quantitative measure of the amount of type IIcollagen degraded.

Collagen type II is present in hyaline cartilage of joints in the adultskeleton. Quantitation of the collagen type II telopeptides in a bodyfluid, for example by way of a monoclonal antibody that recognizes anepitope in the peptide structure, would provide a quantitative measureof whole-body cartilage destruction or remodeling. In a preferredembodiment, the present invention involves an assay for cartilage tissuedegradation in humans based on quantifying the urinary excretion rate ofat least one member of this family of telopeptides. Such an assay couldbe used, for example, to:

(1) screen adult human subjects for those individuals having abnormallyhigh rates of cartilage destruction as an early diagnostic indicator ofosteoarthritis;

(2) monitor the effects of potential antiarthritic drugs on cartilagemetabolism in osteoarthritic and rheumatoid arthritic patients; or

(3) monitor the progress of degenerative joint disease in patients withosteoarthritis and rheumatoid arthritis and their responses to varioustherapeutic interventions.

Osteoarthritis is a degenerative disease of the articulating cartilagesof joints. In its early stages it is largely non-inflammatory (i.e.distinct from rheumatoid arthritis). It is not a single disease butrepresents the later stages of joint failure that may result fromvarious factors (e.g. genetic predisposition, mechanical overusage,joint malformation or a prior injury, etc.). Destruction of jointarticular cartilage is the central progressive feature ofosteoarthritis. The incidence of osteoarthritis, based on radiographicsurveys, ranges from 4% in the 18-24 year age group to 85% in the 75-79year age group. At present the disease can only be diagnosed by pain andradiographic or other imaging signs of advanced cartilage erosion.

The assays disclosed above may be used to detect early evidence ofaccelerated cartilage degradation in mildly symptomatic patients, tomonitor disease progress in more advanced patients, and as a means ofmonitoring the effects of drugs or other therapies.

In normal young adults (with mature skeletons) there is probably verylittle degradation of cartilage collagen. A test that could measurefragments of cartilage collagen in the urine (and in the blood and jointfluid) would be very useful for judging the "health" of cartilage in thewhole body and in individual joints. The type II collagen-specificpeptide assays described above will accomplish this. In the long term,such an assay could become a routine diagnostic screen for spottingthose individuals whose joints are wearing away. They could be targetedearly on for preventative therapy, for example, by the next generationof so-called chondroprotective drugs now being evaluated by the majorpharmaceutical companies who are all actively seeking better agents totreat osteoarthritis.

Other diseases in which joint cartilage is destroyed include: rheumatoidarthritis, juvenile rheumatoid arthritis, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, thelow back pain syndrome, and other infectious forms of arthritis. Thetype II collagen-specific assays described herein could be used todiagnose and monitor these diseases and evaluate their response totherapy, as disclosed above in connection with osteoarthritis.

Type III Collagen Telopeptides

As pointed out above, human type lII collagen telopeptides that may bepresent in body fluids are expected to have a core structure embodied inthe following parent structures: ##STR12## is hydroxylysyl pyridinoline.

By analogy to the type II peptides, the type III collagen peptides mayoccur in glycosylated forms of the core structure. For example,galactose residues or glucosylgalactose residues may be attached to thecore structure, e.g. by way of hydroxyl groups.

The cross-linking residue of the type III collagen peptides is depictedas a 3-hydroxypyridinium residue, hydroxylysyl pyridinoline. The type IItelopeptide structures have been found to primarily have hydroxylysylpyridinoline cross-linking residues. However, whereas the type IIcollagen peptides are derived from the C-terminal telopeptide region oftype II collagen, the type III collagen peptides may be derived fromeither the N-terminal or the C-terminal of type III collagen, as long asat least one cross-linking residue is present.

Type III collagen is present in many connective tissues in associationwith type I collagen. It is especially concentrated in vascular walls,in the skin and in, for example, the synovial membranes of joints whereits accelerated turnover might be observed in inflammatory jointdiseases such as rheumatoid arthritis.

A specific assay for type III collagen degradation by quantitatingcross-linked type III collagen peptides as diagnosed above, can be usedfor detecting, diagnosing, and monitoring various inflammatorydisorders, possibly with particular application to the vasculitissyndromes. In conjunction with assays for measuring bone type I andcartilage type II collagen degradation rates, such an assay could beused as a differential diagnostic tool for patients with variousdegenerative and inflammatory disorders that result in connective tissuedestruction or pathological processes.

Isolation of Type II and Type III Collagen Telopeptides

General Procedure:

Urine is collected form a normal adolescent during a rapid phase ofskeletal growth. Using a sequence of chromatographic steps that includebut are not limited to, adsorption on selective cartridges of ahydrophobic interaction support and an ion-exchange support andmolecular sieve, ion-exchange and reverse-phase HPLC columnchromatography steps, individual peptides are isolated. The cross-linkedpeptides containing HP (and LP) residues are detected during columnchromatography by their natural fluorescence (Ex max 297 nm <pH 4, Exmax 330 nm, >pH 6; Em max 390 nm). An exemplary isolation procedure isprovided in the Example below.

Specific Example:

Fresh urine (at 4° C.) diluted 5 times with water and adjusted to 2%(v/v) trifluoroacetic acid, passed through a C-18 hydrophobic bindingcartridge (Waters C-18 Sep-pak prewetted with 80% (v/v) acetonitrilethen washed with water). Retained peptides were washed with water theneluted with 3 ml of 20% (v/v) acetonitrile, and this eluent was adjustedto 0.05M NH₄ HCO₃, 10% (v/v) acetonitrile by addition of an equal volumeof 0.1M NH₄ HCO₃. This solution was passed through a QMA-Sep-pak(Waters), which was washed with 10 ml of 0.1M NaCl, 20% (v/v)acetonitrile followed by 10 ml of water and the peptides were theneluted with 3 ml of 1% (v/v) trifluoroacetic acid and dried by Speed-Vac(Savant).

Peptides were fractionated in three chromatographic steps. The firststep was molecular sieve chromatography on a column of Bio-Gel P-10 (BioRad Labs, 2.5 cm×90 cm) eluted by 10% (v/v) acetic acid, monitoring theeffluent for HP fluorescence as shown in FIG. 2. In FIG. 2, the Y-axisis the relative fluorescence emission at 390 nm (297 nm excitation), andthe X-axis is the fraction number. The fraction size was 4 ml. Thefractions indicated as II are enriched in the cross-linked collagen typeII telopeptides. The cross-linked collagen type I telopeptides arecontained in the fractions indicated as III and IV. Fractions spanningpool II (enriched in the type II collagen cross-linked peptides) werecombined, freeze-dried and fractionated by ion-exchange columnchromatography on a DEAE-HPLC column (TSK-DEAE-5PW, 7.5 mm×7.5 mm,Bio-Rad Labs), equilibrated with 0.02M Tris/HCl, 10% (v/v) acetonitrile,pH 7.5 and eluted with a gradient of 0-0.5M NaCl in the same buffer asshown in FIG. 2.

FIG. 3A plots relative fluorescence emission at 390 nm (330 nmexcitation) versus elution time. The cross-linked collagen type IItelopeptides are found primarily in the segment indicated as IV. FIG. 3Bplots absorbance at 220 nm as a function of elution time in minutes.Pool IV contains the type II collagen cross-linked peptides. Individualpeptides were then resolved from pool IV by reverse phase HPLC on a C-18column (Aquapore RP-300, 25 cm×4.6 mm, Brownlee Labs), eluting with agradient of 0-30% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid.FIG. 4A shows a plot of relative fluorescence intensity at 390 nm (297nm excitation) as a function of elution time. The peaks associated withparticular peptides are indicated in FIG. 4A. FIG. 4B shows the relativeabsorbance at 220 nm as a function of time.

Cross-linked peptide fragments of type III collagen containing HPcross-linking residues may be isolated by a similar combination of stepsfrom the urine of normal growing subjects or, for example, from theurine of patients with inflammatory disorders of the vasculature.

Type I Collagen Telopeptides

This aspect of the invention is based on the discovery that both lysylpyridinoline (LP) and hydroxylysyl pyridinoline (HP) peptide fragments(i.e., telopeptides, as used herein) derived from reabsorbed bonecollagen are excreted in the urine without being metabolized. Theinvention is also based on the discovery that no other connectivetissues contain significant levels of LP and that the ratio of HP to LPin mature bone collagen remains relatively constant over a person'slifetime.

FIG. 5 compares the concentration of HP and LP in both cortical andcancellous human bone with age. It is observed that the concentration ofHP plus LP cross-links in bone collagen reaches a maximum by age 10 to15 years and remains reasonably constant throughout adult life.Furthermore, the ratio of HP to LP, shown in FIG. 6, shows little changethroughout life, remaining constant at about 3.5 to 1. These baselinedata demonstrate that the 3-hydroxypyridinium cross-links in bonecollagen remains relatively constant and therefore that body fluidsderived from bone collagen degradation will contain 3-hydroxypyridiniumcross-linked peptide fragments at concentrations proportional to theabsolute rate of bone resorption.

Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen,the method for determining the absolute rate of bone resorption, in itssimplest form, is based on quantitating the concentration of peptidefragments containing 3-hydroxypyridinium cross-links and preferablylysyl pyridinoline (LP) cross-links in a body fluid.

As used in this description and in the appended claims with respect totype I, II, or III telopeptides, by "quantitating" is meant measuring byany suitable means, including but not limited to spectrophotometric,gravimetric, volumetric, coulometric, immunometric, potentiometric, oramperometric means the concentration of peptide fragments containing3-hydroxypyridinium cross-links in an aliquot of a body fluid. Suitablebody fluids include urine, serum, and synovial fluid. The preferred bodyfluid is urine.

Since the concentration of urinary peptides will decrease as the volumeof urine increases, it is further preferred that when urine is the bodyfluid selected, the aliquot asseyed be from a combined pool of urinecollected over a fixed period of time, for example, 24 hours. In thisway, the absolute rate of bone resorption or collagen degradation iscalculated for a 24 hour period. Alternatively, urinary peptides may bemeasured as a ratio relative to a marker substance found in urine suchas creatinine. In this way the urinary index of collagen degradation andbone resorption would remain independent of urine volume.

In one embodiment of the present invention, monoclonal or polyclonalantibodies are produced which are specific to the peptide fragmentscontaining lysyl pyridinoline cross-links found in a body fluid such asurine. Type I telopeptide fragments may be isolated from a body fluid ofany patient, however, it is preferred that these peptides are isolatedfrom patients with Paget's disease or from rapidly growing adolescents,due to their high concentration of type I peptide fragments. Type II andtype III telopeptides may be isolated from a body fluid of any patientbut may be more easily obtained from patients suffering from diseasesinvolving type II or type III collagen degradation or from rapidlygrowing adolescents.

Isolation of Type I Collagen Telopeptides

Urine from patients with active Paget's disease is dialyzed in reducedporosity dialysis tubing (<3,500 mol. wt. cut off Spectropore) at 4° C.for 48 h to remove bulk solutes. Under these conditions the peptides ofinterest are largely retained. The freeze-dried non-diffusate is theneluted (200 mg aliquots) from a column (90 cm×2.5 cm) of Bio-Gel P2(200-400 mesh) in 10% acetic acid at room temperature. A region ofeffluent that combines the cross-linked peptides is defined by measuringthe fluorescence of collected fractions at 297 nm excitation/395 nmemission, and this pool is freeze-dried. Further resolution of thismaterial is obtained on a column of Bio-Gel P-4 (200-400 mesh, 90 cm×2.5cm) eluted in 10% acetic acid.

Two contiguous fraction pools are defined by monitoring the fluorescenceof the eluant above. The earlier fraction is enriched in peptidefragments having two amino acid sequences that derive from theC-terminal telopeptide domain of the αI(I) chain of bone type I collagenlinked to a third sequence derived from the triple-helical body of bonetype I collagen. These three peptide sequences are cross-linked with3-hydroxypyridinium. The overlapping later fraction is enriched inpeptide fragments having an amino acid sequence that is derived from theN-terminal telopeptide domain of bone type I collagen linked through a3-hydroxypyridinium cross-links.

Individual peptides are then resolved from each of the two fractionsobtained above by ion-exchange HPLC on a TSK DEAE-5-PW column (Bio Rad7.5 cm×7.5 mm) eluting with a gradient of NaCl (0-0.2M) in 0.02MTris-HCl, pH 7.5 containing 10% (v/v) acetonitrile. The N-terminaltelopeptide-based and C-terminal telopeptide-based cross-linked peptideselute in a series of 3-4 peaks of fluorescence between 0.08M and 0.15MNaCl. The C-terminal telopeptide-based cross-linked peptides elute firstas a series of fluorescent peaks, and the major and minor N-terminaltelopeptide-based cross-linked peptides elute towards the end of thegradient as characteristic peaks. Each of these is collected,freeze-dried and chromatographed on a C-18 reverse phase HPLC column(Vydac 218TP54, 25 cm×4.6 mm) eluted with a gradient (0-10%) ofacetonitrile: n-propanol (3:1 v/v) in 0.01M trifluoroacetic acid. About100-500 μg of individual peptide fragments containing3-hydroxypyridinium cross-links can be isolated by this procedure from asingle 24 h collection of Paget's urine.

Amino acid compositions of the major isolated peptides confirmed purityand molecular sizes by the whole number stoichiometry of recovered aminoacids. N-terminal sequence analysis by Edman degradation confirmed thebasic core structures corresponding to the sequences of the knowncross-linking sites in type I collagen and from the matching amino acidcompositions. The N-terminal telopeptide sequence of the α2(I) chain wasblocked from sequencing analysis due presumably to the known cyclizationof the N-terminal glutamine to pyrrolidone carboxylic acid.

A typical elution profile of N-terminal telopeptides obtained by theabove procedure is shown in FIG. 7A. The major peptide fragment obtainedhas an amino acid composition: (Asx)₂ (Glx)₂ (Gly)₅ Val-Tyr-Ser-Thr,where Asx is the amino acid Asp or Asn and Glx is the amino acid Gln orGlu. The sequence of this peptide is represented by Formula III below.

The C-terminal telopeptide-based cross-linked peptides resolved byreverse phase HPLC as described above are shown in FIG. 7B. As can beseen from this figure, these peptides are further resolved into a seriesof C-terminal telopeptides each containing the 3-hydroxypyridiniumcross-links. The major peptide, shown in FIG. 7B, was analyzed asdescribed above and was found to have the amino acid composition: (Asp)₅(Glu)₄ (Gly)₁₀ (His)₂ (Arg)₂ (Hyp)₂ (Ala)₅. The sequence of this peptideis represented by formula IV below. It is believed that the otherC-terminal telopeptide-based cross-linked peptides appearing as minorpeaks in FIG. 7B represent additions and deletions of amino acids to thestructure shown in Formula IV. Any of the peptides contained withinthese minor peaks are suitable for use as immunogens as described below.##STR13## represents the HP or LP cross-links and Gln representsglutamine or pyrrolidone carboxylic acid.

Equivalents of the peptides represented by the above structures includethose cases where there is some variation in the peptide structure.Examples of such variation include 1-3 amino acid additions to the N andC termini as well as 1-3 terminal amino acid deletions. Smaller peptidefragments of the molecule represented by Formula IV derived from boneresorption are especially evident in urine. These are found in the minorpeaks of the C-terminal telopeptide fraction seen in FIG. 7B and can beidentified by amino acid composition and sequence analysis,

Examples of Procedures for Quantitating Peptides

A. Immunological Procedure For Quantitating Peptides

Immunological binding partners capable of specifically binding topeptide fragments derived from bone collagen obtained from aphysiological fluid can be prepared by methods well known in the art.The preferred method for isolating these peptide fragments is describedabove. By immunological binding partners as used herein is meantantibodies and antibody fragments capable of binding to a telopeptide,

Both monoclonal and polyclonal antibodies specifically binding thepeptides disclosed herein and their equivalents are prepared by methodsknown in the art. For example, Campbell, A. M. Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13 (1986). Elsevier, hereinincorporated by reference. It is possible to produce antibodies to theabove peptides or their equivalents as isolated. However, because themolecular weights of these peptide fragments are generally less than5,000, it is preferred that the hapten be conjugated to a carriermolecule. Suitable carrier molecules include, but are not limited to,bovine serum albumin, ovalbumin, thyroglobulin, and keyhole limpethemocyanin (KLH). Preferred carriers are thyroglobulin and KLH.

It is well known in the art that the orientation of the hapten, as it isbound to the carrier protein, is of critical importance to thespecificity of the antiserum. Furthermore, not all hapten-proteinconjugates are equally successful immunogens. The selection of aprotocol for binding the particular hapten to the carrier proteintherefore depends on the amino acid sequence of the urinary peptidefragments selected. For example, if the peptide represented by FormulaIII is selected, a preferred protocol involves coupling this hapten tokeyhole limpet hemocyanin (KLH), or other suitable carrier, withglutaraldehyde. An alternative protocol is to couple the peptides to KLHwith a carbodiimide. These protocols help to ensure that the preferredepitope, namely Tyr and a 3-hydroxy-pyridinium cross-link, are presentedto the primed vertebrate antibody producing cells (e.g., B lymphocytes).

Other peptides, depending on the source, may require different bindingprotocols. Accordingly, a number of binding agents may be suitablyemployed. These include, but are not limited to, carbodiimides,glutaraldehyde, mixed anhydrides, as well as both homobifunctional andheterobifunctional reagents (see for example the Pierce 1986-87 catalog,Pierce Chemical Co., Rockford, Ill. Preferred binding agents includecarbodiimides and heterobifunctional reagents such asm-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS).

Methods for binding the hapten to the carrier molecule are known in theart. See for example, Chard, T., Laboratory Techniques in Biochemistryand Molecular Biology, Vol. 6 (1987) Partz Elsevier, N.Y., hereinincorporated by reference.

Either monoclonal or polyclonal antibodies to the hapten-carriermolecule immunogen can be produced. However, it is preferred thatmonoclonal antibodies (MAD) be prepared. For this reason it is preferredthat immunization be carried out in the mouse. Immunization protocolsfor the mouse usually include an adjuvant. Examples of suitableprotocols are described by Chard, T. (1987) vida supra. Spleen cellsfrom the immunized mouse are harvested and homogenized and thereafterfused with cancer cells in the presence of polyethylene glycol toproduce a fused cell hybrid which produces monoclonal antibodiesspecific to peptide fragments derived from collagen. Examples of suchpeptides are represented by the formulas given above. Suitable cancercells include myeloma, hepatoma, carcinoma, and sarcoma cells. Detaileddescriptions of this procedure, including screening protocols, protocolsfor growing selected hybrid cells and harvesting monoclonal antibodiesproduced by the selected hybrid cells are provided in Galfre, G. andMilstein, C., Meth. Enzymol., 73:1 (1981). A preferred preliminaryscreening protocol involves the use of peptide fragments derived frombone collagen resorption and containing 3-hydroxypyridinium cross-linksin a solid phase radioimmunoassay.

The monoclonal antibodies or other immunological binding partners usedin connection with the present are preferably specific for a particulartype of collagen telopeptide. For example, assays for the type II ortype III collagen degradation telopeptides should preferably be able todistinguish between the type I, type II, and type III peptides. However,in some cases, such selectivity will not be necessary, for example, ifit is known that a patient is not suffering degradation of one type ofcollagen but is suspected of suffering degradation from the assayed typeof collagen. Because of the differences in amino acid sequences betweenthe type I, type II, and type III families of telopeptides,cross-reactivity should not occur to a significant degree. Indeed,hybridomas can be selected for during the screening of splenocyte fusionclones that produce monoclonal antibodies specific for the cross-linkedtelopeptide of interest (and lack affinity for those of the other twocollagen types). Based on the differences in sequence of the isolatedpeptide structures, such specificity is entirely feasible. Peptidefragments of the parent types I, II and III collagens, suitable for suchhybridoma screening, can be prepared from human bone, cartilage andother tissues and used to screen clones from mice immunizedappropriately with the individual cross-linked peptide antigens isolatedfrom body fluid.

Immunological binding partners, especially monoclonal antibodies,produced by the above procedures, or equivalent procedures, are employedin various immunometric assays to quantitate the concentration of thepeptides having 3-hydroxypyridinium cross-links described above. Theseimmunometric assays comprise a monoclonal antibody or antibody fragmentcoupled to a detectable marker. Examples of suitable detectable markersinclude but are not limited to: enzymes, coenzymes, enzyme inhibitors,chromophores, fluorophores, chemiluminescent materials, paramagneticmetals, spin labels, and radionuclides. Examples of standardimmunometric methods suitable for quantitating the telopeptides include,but are not limited to, enzyme linked immunosorbent assay (ELISA)(Ingvall, E., Meth. Enzymol., 70 (1981)), radio-immunoassay (RIA), and"sandwich" immunoradiometric assay (IRMA).

In its simplest form, these immunometric methods can be used todetermine the absolute rate of bone resorption or collagen degradationby simply contacting a body fluid with the immunological binding partnerspecific to a collagen telopeptide having a 3-hydroxypyridiniumcross-link.

It is preferred that the immunometric assays described above beconducted directly on untreated body fluids (e.g. urine, blood, serum,or synovial fluid). Occasionally, however, contaminating substances mayinterfere with the assay necessitating partial purification of the bodyfluid. Partial purification procedures include, but are not limited to,cartridge adsorption and elution, molecular sieve chromatography,dialysis, ion exchange, alumina chromatography, hydroxyapatitechromatography and combinations thereof.

Test kits, suitable for use in accordance with the present invention,contain monoclonal antibodies prepared as described above thatspecifically bind to peptide fragments having 3-hydroxypyridiniumcross-links derived from collagen degradation found in a body fluid. Itis preferred that the monoclonal antibodies of this test kit be coupledto a detectable marker of the type described above. Test kits containinga panel of two or more immunological binding partners are alsocontemplated. Each immunological binding partner in such a test kit willpreferably not cross-react substantially with another type oftelopeptide. For example, an immunological binding partner that bindsspecifically with a type II collagen telopeptide should preferably notcross-react with either a type I or type III collagen telopeptide. Asmall degree (e.g. 5-10%) of cross-reactivity may be tolerable.

B. Electrochemical Procedure For Assaying For Peptides

An alternative procedure for assaying for the above-described peptidesconsists of measuring a physical property of the peptides having3-hydroxypyridinium cross-links. One such physical property relies uponelectrochemical detection. This method consists of injecting an aliquotof a body fluid, such as urine, into an electrochemical detector poisedat a redox potential suitable for detection of peptides containing the3-hydroxypyridinium ring. The 3-hydroxypyridinium ring, being a phenol,is subject to reversible oxidation and therefore the electrochemicaldetector (e.g., Model 5100A Coulochem sold by Esa 45 Wiggins Ave.,Bedford, Mass. is a highly desirable instrument suitable forquantitating the concentration of the present peptides. Two basic formsof electrochemical detector are currently commercially available:amperometric (e.g., BioAnalytical Systems) and coulometric (ESA, Inc.,Bedford, Mass. 01730). Both are suitable for use in accordance with thepresent invention, however, the latter system is inherently moresensitive and therefore preferred since complete oxidation or reductionof the analyzed molecule in the column effluent is achieved. Inaddition, screening or guard electrodes can be placed "upstream" fromthe analytical electrode to selectively oxidize or reduce interferingsubstances thereby greatly improving selectivity. Essentially, thevoltage of the analytical electrode is tuned to the redox potential ofthe sample molecule, and one or more pretreatment cells are set todestroy interferents in the sample.

In a preferred assay method, a standard current/voltage curve isestablished for standard peptides containing lysyl pyridinoline orhydroxylysyl pyridinoline in order to determine the proper voltage toset for optimal sensitivity. This voltage is then modified dependingupon the body fluid, to minimize interference from contaminants andoptimize sensitivity. Electrochemical detectors, and the optimumconditions for their use are known to those skilled in the art. Complexmixtures of body fluids can often be directly analyzed with theelectrochemical detector without interference. Accordingly, for mostpatients no pretreatment of the body fluid is necessary. In some caseshowever, interfering compounds may reduce the reliability of themeasurements. In such cases, pretreatment of the body fluid (e.g.,urine) may be necessary.

Accordingly, in an alternative embodiment of the invention, a body fluidis first purified prior to electrochemically titrating the purifiedpeptide fragments. The purification step may be conducted in a varietyof ways including but not limited to dialysis, ion exchangechromatography, alumina chromatography, hydroxyapatite chromatography,molecular sieve chromatography, or combinations thereof. In a preferredpurification protocol, a measured aliquot (25 ml) of a 24 hour urinesample is dialyzed in reduced porosity dialysis tubing to remove thebulk of contaminating fluorescent solutes. The non-diffusate is thenlyophilized, redissolved in 1% heptafluorobutyric acid (HFBA), an ionpairing solution, and the peptides adsorbed on a Waters Sep-Pak C-18cartridge. This cartridge is then washed with 5 ml of 1% HFBA, and theneluted with 3 ml of 50% methanol in 1% HFBA.

Another preferred method of purification consists of adsorbing ameasured aliquot of urine onto an ion-exchange adsorption filter andeluting the adsorption filter with a buffered eluting solution. Theeluate fractions containing peptide fragments having 3-hydroxypyridiniumcross-links are then collected to be assayed.

Still another preferred method of purification employs molecular sievechromatography. For example, an aliquot of urine is applied to a Bio-GelP2 or Sephadex G-20 column and the fraction eluting in the 1000-5000Dalton range is collected. It will be obvious to those skilled in theart that a combination of the above methods may be used to purify orpartially purify urine or other body fluids in order to isolate thepeptide fragments having 3-hydroxypyridinium cross-links. The purifiedor partially purified peptide fragments obtained by the above proceduresmay be subjected to additional purification procedures, furtherprocessed or assayed directly in the partially purified state.Additional purification procedures include resolving partially purifiedpeptide fragments employing high performance liquid chromatography(HPLC) or microbore HPLC when increased sensitivity is desired. Thesepeptides may then be quantitated by electrochemical titration.

A preferred electrochemical titration protocol consists of tuning theredox potential of the detecting cell of the electrochemical detector(Coulochem Model 5100A) for maximum signal with pure HP. The detector isthen used to monitor the effluent from a C-18 HPLC column used toresolve the partially purified peptides.

C. Fluorometric Procedure For Quantitating Peptides

An alternative preferred method for quantitating the concentration ofpeptides having 3-hydroxypyridinium cross-links as described herein isto measure the characteristic natural fluorescence of these peptides.For those body fluids containing few naturally occurring fluorescentmaterials other than the 3-hydroxypyridinium cross-links, fluorometricassay may be conducted directly without further purification of the bodyfluid. In this case, the peptides are resolved by HPLC and the naturalfluorescence of the HP and LP amino acid residues is measured at 395 nmupon excitation at 297 nm, essentially as described by Eyre, D. R., etal., Analyte. Biochem. 137:380 (1984), herein incorporated by reference.

It is preferred, in accordance with the present invention, that thefluorometric assay be conducted on urine. Urine, however, usuallycontains substantial amounts of naturally occurring fluorescentcontaminants that must be removed prior to conducting the fluorometricassay. Accordingly, urine samples are first partially purified asdescribed above for electrochemical detection. This partially purifiedurine sample can then be fluorometrically asseyed as described above.Alternatively, the HP and LP cross-linked peptides in the partiallypurified urine samples or other body fluids can be hydrolyzed in 6M HClat about 108° C. for approximately 24 hours as described by Eyre, et al.(1984) vida supra. This process hydrolyzes the amino acids connected tothe lysine precursors of "tripeptide" HP and LP cross-links, producingthe free HP and LP amino acids represented by Formulae I and II. Thesesmall "tripeptides" are then resolved by the techniques described above,preferably by HPLC, and the natural fluorescence is measured (Ex 297 nm,Ex 390 nm).

Optionally, the body fluid (preferably urine) is passed directly througha C-18 reverse phase affinity cartridge after addingacetonitrile/methanol 5 to 10% V/V. The non-retentate is adjusted to0.05-0.10M with a cationic ion-pairing agent such as tetrabutyl ammoniumhydroxide and passed through a second C-18 reverse phase cartridge. Thewashed retentate, containing fluorescent peptides, from this secondcartridge is eluted with acetonitrile:water (or methanol:water), driedand fluorescent peptides are analyzed by reverse phase HPLC or microboreHPLC using an anionic ion-pairing agent such as 0.01M trifluoroaceticacid in the eluant.

FIG. 8A displays the elution profile resolved by reverse phase HPLC ofnatural fluorescence for a hydrolysate of peptide fragments from normalhuman urine. Measurement of the integrated area within the envelope of agiven component is used to determine the concentration of that componentwithin the sample. The ratio of HP:LP found in normal human urine andurine from patients having Paget's disease, FIG. 8B, are bothapproximately 4.5:1. This is slightly higher than the 4:1 ratio found inbone itself (Eyre, et al., 1984). The higher ratio found in urineindicates that a portion of the HP fraction in urine may come fromsources other than bone, such as the diet, or other sources of collagendegradation, i.e., cartilage catabolism. It is for this reason that itis preferred that LP which derives only from bone be used to provide anabsolute index of bone resorption. However, in the absence of excessivecartilage degradation such as in rheumatoid arthritis or in cases wherebone is rapidly being absorbed, HP or a combination of HP plus LP may beused as an index of bone resorption.

While the invention has been described in conjunction with preferredembodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and alterations to the subject matter set forth herein.Hence, the invention can be practiced in ways other than thosespecifically described herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of analyzing abody fluid sample for the presence of an analyte indicative of type Icollagen degradation in vivo, comprising the steps of contacting thebody fluid sample with an immunological binding partner which is capableof binding to the analyte, detecting any binding of the immunologicalbinding partner in the body fluid sample, and correlating the detectedbinding to type I collagen degradation in vivo, wherein theimmunological binding partner is capable of binding to a peptidecontaining a 3-hydroxypyridinium cross-link derived from thecarboxy-terminal telopeptide domain of type I collagen, the peptidecomprising ##STR14## wherein K--K--K is a 3-hydroxypyridinium cross-linkselected from among hydroxylysyl pyridinoline and lysyl pyridinoline. 2.The method of claim 1, wherein the analyte is further characterized byhaving a molecular weight less than 5,000.